JPS6296087A - Nodd related gene of rizobium japonica 191 - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] The box-related genes of USD81 to 1 are [l5DA 1
The EcoRI digest of 91-derived genome ON was added to Rhizobium melilote 4 (Rhizobium mel
pRMS containing all of nod A, nod B, and part of nod D and nod C of
It was isolated by hybridizing with L42 and examining it (Egelhoff (1985)). Two strongly hybridized fragments were found, one approximately 6 Kb.
p.

The other ones were approximately 3 Kbp and were named EA14-17 and EA4-19, respectively. Fragment EA14-17 contains goD-rl and fragment EA4-19 contains nodD
Contains r2. Neither the 6Kbp nor the 3Kbp fragments contained sequences homologous to box A, block B, or nodC. nodA and/or nod
The sequences related to B and the sequences related to nod C are
It was identified on a separate 9.6 Kbp EcoRr fragment. Therefore, the genetic makeup of USDA 191 is different from that of previously reported strains.
nodD-related sequences are nod A and nod B
.

They differ in that they are separated by at least a few kilobases from the nod C position and also from each other.

(Margins below) Figure 1 shows a partial restriction map of the two Eco R1 fragments. The majority of each was sequenced, including all of the nodD-related coding region. In the 3kb Eco R1 fragment, 2886 base pairs were sequenced corresponding to almost the entire fragment. The shaded area is 36, 39
n which encodes a protein of 321 amino acids with a molecular weight of 3
The position of the od D-r2 oven reading frame is shown graphically. Arrows indicate n with respect to the restriction map of the 3 kb fragment.
The transcription direction of odD-r2 is shown. The sequence of 2543 base pairs was determined for the 6kb fragment. It encodes a 312 amino acid fluorescent protein with a molecular weight of 35,300 spanning the open reading frame and the coding region of EA14-17. Arrows indicate transcription direction with respect to the restriction map. Table 1 shows the code sequence.

356 bases of 5' untranslated sequence including promoter.

and nodD − containing 82 bases of 3′ untranslated sequence.
The DNA sequence of r2 is shown. Table 2 contains 337 base pairs of 5' untranslated sequence including the promoter coding sequence.

and nod, which contains a 128 base pair 3° untranslated region.
Contains the nucleotide sequence of D-rl. Table 3 shows the amino acid sequences encoded by nod D-r2 and nod D-rl, respectively. The deduced amino acid sequences of nodD-r2 and nodD-rl show approximately 70% homology, and each of them has a similar amino acid sequence to that deduced from the sequence of the nodD gene of B. meliloti. It showed approximately 70% homology. Based on the functional differences listed below.

The degree of non-homology appears to be sufficient to give rise to significant functional dissimilarity.

The functional characteristics of the nod D-related genes of tlsD^191 are listed below. Mutants in nod D-rl are unable to form soybean root nodules quickly and effectively; soybean root nodule formation is delayed, and the nodules that do form have poor ability to fix nitrogen.

It is approximately 174 times the capacity of a normal mature root nodule. Vector pR
USD containing extra copies of the gadfly D-rl on K290
The amount of exopolysaccharide formed by the structure of A191 was determined by the amount of exopolysaccharide formed by the structure of wild-type USDA19 in several species.
substantially less than that of 1. nodD-rl is expressed in free-living bacterial cells. The nodD-rl on the broad host range plasmid is derived from Zobium trifolii (R,tri
folii) It is not possible to restore the nodule-forming ability to clover to the nod D-mutant.

The USDA 191 nod D-r2 mutant, compared to the wild type and the nod D-rl mutant,
Tropical legume 5iratr
o) It is not possible to form a root nodule. Vector pRK
The structure of tlsDA 191 containing an extra copy of nodD-r2 present on 290 does not produce any appreciable change in exopolysaccharide production, as judged by colony morphology. Compared to nodD-rl, nodD-r2 on the broad host range plasmid complements and forms root nodules in clovers when transferred to the Rhizobium trifolii (R, trifolii) nodD-mutant. It is possible to recover Koto's abilities.

By transferring both the nod D-related genes of USDA 191 to Rhizobium meliloti (R, meliloti) or Rhizobium leguminosarum (R) (along with other genes contained in USDA 191 S) ), such bacterial cells will not form normal soybean root nodules. The root nodules produced instead of the above are defective, lack nitrogen fixation ability, do not contain leghemoglobin, nodulin 35 or nodulin 29, and are free of such defects. It is impossible to extract viable bacteria from the root nodule.The R. meliloti host was a wild-type strain with respect to its own symbiotic genes, whereas the R. meliloti host was wild-type with respect to its own symbiotic genes.
leguminosarum (L area and m1no and deaf ridge strain) lacked its own symbiotic gene.Contrary to the above, ANU2 of USDA191'L plasmid containing one nod D-related gene
ANU 265/pSy
The transconjugant of m191 is able to form normal nitrogen-fixing root nodules in soybean.

2 (7) Comparison of the amino acid sequences encoded by LISDA 191nod D-related genes with amino acid sequences in protein sequence databases revealed unexpected homology between these genes and the n>R genes of E and codan. The gender has been revealed. The function of hLR is that of a regulator regarding the transcription of genes involved in the biosynthetic process of amino acid lysine. Furthermore, hLR exerts feedback control over its own expression (P, Stragieret
a1, (1983) J, Mo1. Bio1,
168:333). Furthermore.

b! Since R controls expansion A in response to diaminopimelic acid, a bacterial cell wall component, μpd-related genes are linked to cell wall components or Rhizobium.
um) Rhizobium (Rhizobiu) during soybean interaction
m) or can also mediate the expression of other nod genes in response to other substances released either by the soybean. Although the above observations suggest a regulatory function for the nod D-related genes in USDA 191, the present invention does not rely on the functional theory for the effective functioning of the nod D-related genes.

NodD-related genes nodDrl and nodD-r2 of cloned tlsDA 191 are U
SDA 191 and other soybean root-knot bacteria are useful for genetically manipulating parameters such as rate, efficiency, comparability and agronomic specificity of soybean root-knot formation. Furthermore, nod D-rl helps regulate exopolysaccharide production by cultivated Rhizobium strains, and the properties of this strain make exopolysaccharide synthesis more difficult than commercial Rhizobium production methods. Because it limits productivity and acts as an inhibiting factor for isolation techniques for enrichment and purification of bacterial cells from cultivation media.

It is an important presence. A number of genetic manipulations known in the art include
Used to alter the amount of genes, the rate and timing of gene expression, or to modulate gene expression by environmental factors. These techniques include, but are not limited to, expression of one or both of the genes under the control of other promoters, altering the amount of the genes in the host cell.

This includes enabling inducible or constitutive expression of one or more gene copies and forming translational fusions that combine the functions of related genes with each other or with other genes. rate and timing of expression, and up-”
Other techniques of genetic manipulation that provide conditions for the 54 or down sm clauses may be used, as will be understood by those skilled in the art. The disclosed nucleotide sequence data may be modified using known DNA sequence modification techniques such as, but not limited to, the use of restriction endonucleases, ligases, exonucleases, and site-directed mutagenesis and chemical DNA synthesis. Using techniques that involve processes.

Provides a means for forming the described gene structures.

The promoters of nodD-rl and nodD-r2.

its normally associated nodD-rl and nodD-
It is useful for expressing coding sequences other than the r2 structural gene. Such coding sequences (herein referred to as exogenous coding sequences) may be derived from other gene 9 synthetic NAs of the same or different organisms or a combination of sources forming chimeric coding sequences, and may be structurally The only limitations are those known in the art for lfl translatable coding sequences. Formation of a composite gene with an exogenous coding sequence and the promoter of nodD-rl or nodD-r2 leads to expression of this composite gene under circumstances characteristic of parietal gyrus-D-rl or nodD-r2 expression. . for example.

The nod D-rl promoter drives expression in both free-living bacteria and soybean plants.
The ID-r2 promoter drives expression in soybean plants and also in free-living Rhizobia.

A promoter is defined herein as a nucleotide sequence upstream of the transcription start site that contains all control regions necessary for transcription. Promoter regions can be identified, in part, by the technique of S1 nuclease noping, which identifies transcription start sites. Further identification of promoters is based on function. Whereas parts of the sequence proximal to the transcription start site are often essential for promoter function;
More distal upstream fragments may function as a "fine-tuning" response to modulators. Certain consensus sequences have been shown to be characteristic of prokaryotic promoters (Rosenberg and Court (1999)).
79)).

The above criteria ensure that nodD-rl and nodD-rl of Rhizobium japonicum (R,D and Sniper μL) USDA 191 are within the 5' untranslated region upstream from the start of the nodDrl or nodD-r2 coding region. It can be used to identify the promoter of nodD-r2. The nodD-rl promoter is approximately 1970
It is located in a sequence extending from position (Table 2) to the transcription start site (a point upstream of the start of translation at position 1663). nodD-
The r2 promoter is located in a sequence extending from approximately position 951 (Table 1) to the transcription site.

The sequence data provided herein, when combined with methods known in the art, will allow one of ordinary skill in the art to construct the desired composite gene whose expression is regulated by -rl or +dD-r2. It is sufficient for Such methods include, but are not limited to, the following:
restriction endonuclease cleavage at desired sites in the sequence;
These include ligation of the desired fragment to other desired DNA fragments, chemical synthesis of oligonucleotides, and site-directed mutagenesis.

It is known that variations in the sequence of promoters and coding sequences can be made without substantially affecting function. The occurrence of certain such variants, known as allelic variants, is limited in frequency in nature.

It is known that for coding sequences, homologues with different nucleotide sequences encoding the same amino acid sequence are possible due to the nature of the genetic code. Other variants affecting the amino acid sequence are also possible, but this nevertheless does not substantially alter the functional properties. For a promoter, some parts of the sequence may be essential, whereas other parts may be allowed to vary without affecting the quantitative or qualitative functional properties of the promoter. be.

Based on comparisons between promoters and similar functions.

When the overall homology is 90% or more, mutations that do not substantially affect the function of the promoter can be tolerated and identified.

Therefore, the range of compatible equivalents includes, in the case of coding sequences, homologues that encode functionally equivalent proteins, and in the case of promoters, functionally equivalent homologues with at least 90% sequence homology. Contains homologs.

The following examples merely illustrate the invention. Cloning, DNA isolation, amplification and purification, DNA ligase, DNA polymerase, except as noted below.

Standard techniques for enzymatic reactions and various separation techniques, including restriction endonucleases and the like, are known and commonly used by those skilled in the art (eg.

R, Wu + ed. (1979) Meth, E.
zymo1, 68:R, Wu.

et al., eds, (1983) Meth
, Enzymo1, 100, 匪; L, Gros
Sman and K., Moldave, eds.
(1980) Meth.

Enzymo1, 65; J, H, Miller (
1972) Ex eriments in Mole
cular Genetics; Davis, R.
et Dan, (1980) Advanced B
Acterjal Genetics HR, F.

5chleif and P, C, Wensink (
1982) Practical Methods i
n Mo1ecular Red Rainbow ■L; and T.

Manniatis et al. (1982)
Molecular Katanae L).

When abbreviations are used, they are those considered standard in the field and commonly used in specialized journals such as those cited herein. DN
The term "cloned" as used with respect to A sequences refers to a specific DNA sequence that has been isolated and purified substantially free of contaminating DNA. However, cloning D
The NA can be maintained as part of a vector or other replicable entity from which the cloned DNA can be separated by conventional techniques including restriction enzyme digestion and electrophoresis.

[The total genomic DNA of l5DA 191 was digested with RI.

Fractionated by agarose gel electrophoresis. S,L.

Plasmid pRMSL42 obtained from Long et al. was used as a hybridization probe under Southern hybridization conditions to identify sequences homologous to that of the probe. Probe beams nod A and n of Zobium meliloti 4 (R, meliloti)
It contained all of nod B, as well as nod D and part of nod C. 9.6 kb and 6.0 kb respectively
and 3. Okb's three strong hybridizing Ecos
Although the R1 fragment was present in IJSD819,
lsDA 191'' u plasmid (absent in derivatives; 9.6 kb fragment contains nod C and nod A
and/or was found to have sequence homology to nc+d B, but no homology to nod D. However, both the 3.0 kb and 6.0 kb fragments were Rhizobium melilotei (Byu mel 1 lot).
i) It had homology to the nodD region.

The Cosmid Clone Bank is partially Ec.

Genomic USDA 191 DNA digested with R1 was transformed into vector psUP 205 (Simon R, et al.
1, (1983) Biotechnology 1 Near 84). When this bank was screened using the nod-specific fragment of pRMSL42, it was found that there were several clones containing USDA 191 nod-related genes. 3.0kb
Cosmids containing the fragment and its flanking fragments did not contain the 6.0 kb or 9.6 kb fragments. Also 6,
The cosmid containing the 0 kb fragment and its adjacent fragments is 3
．． It did not contain regions of 0 kb or 9.6 kb.

Therefore, the nod D-related sequences at these two sites are separated from each other and at least a few kilobases from the nodA, nodB, and dishLC loci. Furthermore, no mouse LD-related sequences adjacent to dish dA, dish dB, and dish rainbow C were detected.

As a result of further hybridization experiments, the andrd D regions of Rhizobium meliloti (R, meliloti) were found to be 3.0 kb and 6.0 kb of USDA 191. O
It was found that it hybridized only with the central part of the kb fragment.

These two fragments of USDA 191 also hybridized with each other, but only the central portions had homology. The hybridizing region in the center is Maxam, A and G.
ilberj, W. (1977) Proc., N.
Sequenced using at1, Acad, Sci, 74.560 technology. n associated with the 3.0 kb fragment
The od D-related sequence was designated nodD-r2.

Additionally, the dD-related sequences associated with the 6.0 kb fragment were nod
It was called D-rl. 2886 base pairs of the 3.0 kb fragment were sequenced, and 2543 base pairs of the central nod D-related portion of the 6.0 kb fragment were also sequenced. In each case there is a large open reading frame approximately 936 base pairs long in the case of nodD-rl.

Furthermore, in the case of nodD-r2, it was found from the sequence that there is a frame with a length of 963 base pairs. The sequences of EA14-17 and HA4-19, including the open reading frame and 5' and 3' untranslated regions, are shown in Tables 1 and 2. The 300 base pair region preceding the open reading frame (on the 5' side) is.

It contains several regions homologous to the region preceding nodD in Rhizobium meliloti (R. meliloti).

and these areas are 2 nods of USDA 191
It has been identified as a promoter of D-related genes.

USDA containing mutations in each of the nod D-related genes
191 derivatives were made. Insertional mutations were generated by inserting the kanamycin resistance gene into a specific amHI site within the open reading frame of each nod D-related gene. nod D-rl
The Bam-old site of is located in the nucleotide sequence encoding amino acid 135, and the Bam-old site of nodDr2 (
One site is located in the nucleotide sequence encoding amino acid 88. FIG. 1 schematically shows the location of the Bam ) II site. The inserted kanamycin resistance gene is B
Flanked by sequences containing the am 111 site.

In the case of nodDrl, the second mutation is.

It was created by deleting 1.9 kilobases of DNA between the Ban t11 site and the C1a 1 site upstream, and replacing the deleted piece with the kanamycin resistance gene. Mutations in clones were generated by backcrossing to wild-type USDA 191 by double homologous Mi recombination and mutated USDA 191 selected for kanamycin resistance.
The structure of insertion and deletion mutations in the 191 genome was confirmed by plot hybridization analysis.

Insertion and deletion mutations in nodDrl are
The leaky fix-phenotype was fully developed in cultivars Williams and Mu. The 4-week-old plants were slightly pale green, with a color tone intermediate between the green of the normal fix+inoculated plants and the yellow of the non-inoculated plants. Acetylene reducing activity is a measure of nitrogenase activity.

The wild type level was about 174. The number of shared seats was approximately 172, which is the wild type level. In the results of time-course studies, root nodules and nitrogenase activity.

The mutant appeared to be slightly slower than the wild type. In the case of the nod D-rl mutant, the development of root nodules appeared to be delayed by 1-2 days, and the root nodules were not fully developed even after 4-6 weeks. In the case of competitive inoculation, even a slight delay in the application of a strain can lead to significant suppression of that strain in the nodules that form. n
The odD-rl delayed synecdialization mutant exhibits similar behavior when simultaneously present in a mixture of wild-type and mutant bacteria. In this experiment, stationary phase cultures of each strain were mixed at a 10:1 ratio of mutant to wild-type growing cells and inoculated into a soybean cultivar. Soybean plants were grown on vermiculite in Leonardobin. After 4 weeks, the root nodules were harvested from the root surface, the root nodule surface was sterilized, crushed and placed on 9 different selective media. Mutant bacteria were detected in only 12.5% of the root nodules, and all of the root nodules were overgrown with wild-type bacterial cells.

A 6.0 kb fragment containing nodD-rl was cloned into the broad host range vector pRK290 and then [l5DA
191 and transformed. Colonies containing extra copies of nodD-related genes show morphological changes;
Compared to wild type USDA 191, it has a smaller appearance and significantly lower rubber properties. This effect was seen in several different laboratory media. For example, yeast-mantol agar.

Tryptone-yeast extract (Bergerson, F.
J. (ed) (1980) Methods for
h Rainbow ■Two pairs of anus 1■Dansha Demigo Fixaton,
J. Wiley, New York), and a minimal medium containing glycerol as a carbon source and monosodium glutamate as a nitrogen source (Bishop, Dansha, (1976)).
Plant Physiol, 57:542). The morphological changes in the colonies were determined by vector pRK.
USDA 19 containing only 290 or also clones containing insertional mutations in nodD-rl as previously described.
It was not observed in the control strain 1. Morphological changes in colonies were observed in 6. in pRK290. This occurred regardless of the orientation of the Okb fragment. This result supports the conclusion that the effect on colony morphology is due to the presence of the nodD-related gene and that the gene is expressed from its own promoter. This result also indicates that nodDrl is expressed in free living bacterial cells. Changes in colony appearance are interpreted as a decrease in exopolysaccharide synthesis.

The effect of mutations in nodDr2 is nod D -rl
The effect of the mutation was even more subtle. Most strikingly, nodDr2 mutants lacked the ability to form root nodules in the tropical legume sira tro. non-mutated nod D-r2
Extra copies of had no effect on nodule or colony morphology. Unlike the 6.0 kb fragment containing nodDrl,
A 3.0 kb fragment containing non-mutated nodD-r2, R. trifolii no.
It was possible to complement the dD- mutant (recovery of root nodule formation ability). Therefore, Rhizobium trifuori (R
, trifolii) has nod D-r
2, Rhizobium trifolii (Ltrifolii) is siratro (siratro)
tro) cannot form a root nodule. On the other hand, the ability to form root nodules in sira tro is
One of the nodD-related genes in SD81 to 1, especially nod
It is related to the function of the Dr2 gene. Although this result does not exclude the possibility that other genes play a role in these phenomena, it is important to note that the function of EA4-19 does not exclude the possibility that other genes play a role in these phenomena.
This shows that it is different from the i) skin iridescence gene. A 6.0 kb clone containing nodDrl was derived from R. trifolii (R, trifolii) nod.
No complementation of the D-mutant was seen.

General Conclusions As disclosed above, DNA clones encoding USDA 191 nod D-related genes are provided that have unique and distinct functional attributes. This is useful for modifying soybean plate-forming bacteria to improve the timing, efficiency, and competitiveness of the bacteria during the root nodule formation process. As is well known in the art, modifications of coding sequences can be made without altering the fundamental function of the encoded protein. Such modifications may be associated with changes in functional attributes. Also, such modifications do not appear to affect amino acid sequence, translation or transcription efficiency. Furthermore, such modifications are not expected to affect the functional attributes of the genes defined or disclosed herein. This modification is within the scope of modification that would be obvious to one of ordinary skill in the art, and is within the scope of the appended claims, and is considered to be a minor change and no change in value.

The following specimen was deposited with the Agricultural Research Culture Collection (NRRL), Peolia, Illinois, on July 29, 1985.

E. coli carrying clone EA4-19; NR
RL B-15985 clone E. coli carrying EA14-17; NRRL B-15986 (margins below) Table 1 901 TTTTTACCGA CAGATCA
GCT TTGCGCTTGA AGCGCCGC
TT GCCGTCCCCGding CTGGCG[;CG
GGCCCGCGAGGMCGGCGGAAAAGGCG
GCGCCGAAGCCCCTGCA ding ding GTTAG ding GGCAAGGCTGTTGCGGTGGGCGTTG
ACGGT DingTCGCTCCGGCTCGACCCTA
CGGTCGAAAAGACGAAADINGGGCAGG.
AGCGGTCGGCCGTCTTGCCACMAGC
CTGADingCTTCAGGAGGCGCATGDingAAG
AGGGCMCTCCGCGAGCAGAAGCG
C Ding CGCACCCGC AGTCCGCGGM
GCCCTGCTGC ACATCCATCTCT
CCATCATTTCATGGGA dingCCGTTCAA
CCCAGCGCAGTCAGATTCGCAGTTTC
AGGATCATGCGTGGTGATGTCGAT
TCC GATCCTACCAGAAATGTTCA DG CGCACACGCATCCCAGAGCGAAG
CTGTTCGA Ding GAGAGATTCGTG Ding GCG
TGAGTTGCCCAACGAACCAGAAGCT
ACCAGTCGCCGdingGCCGGGTTnACCA
TGATCCCGCCTTTTTTGTCGGGGCAC
TGACCGCA DingAGCGACCCTCCCGTTA
CGACTGGCGATGCACTTCGCAAAAG
CCATTCCCCTGCGGADingCACCGAACDingTCCGCAA CCCArTTTTC CCGC
GTTCAC CGAGGI:TGTC CAGT
GGCCCG CGCCTCACAGCAGTGAT
CCG GCCAGTCTCT GGATGCGCGA
GATATTTCTA CAGGAGGCGT CT
CGCGTTGAATTTCCAATCCGAAAACTT
CGGCGCATGCTCTATCA
ATTGCCDING ACATGCCTCTAAAGCCGA
CGGCGCAACTCGCCACCCTTGAGCC
CGCAACDingGCACGGTCGGCTCAGGGT
CGAGCAGTTingTGGCTGCGGGTTGC23
00Table 2 2000 GAAGAACAGG CTAACC
AAGC CGGAGGATCA CTCCCnA
Ding C GGGGATCAGC TGG Ding CACCG
ACG Ding CAGCTC Ding CGAAAGTGCCGGGGG
TCCGGGTTGCACACCTTTCCATCA
GCACGCTGGAGAGGCCTCTGC MG
CCGAAGT nTGTCCAAC GCACC
ACTCA TTTGGTGTGT CGMTGT
GG Ding GGGA Ding AGTCCCACGCTCTTTAC
GG DING ACTCGGGACGGAGTGCGCTCCA
CTCCCTCAdingGCTCGCTCGCCCCCGC
AG DingCCGCGATGCTCTGCTGCACATC
CAGTTCTCCATCATTTCTTGGGGA Ding A
TGTTTAAC CCAGTTCAG CGCA
GCGACGCTTCAGGATCAGGCT
TTCCG ACGTCATAAdingGCdingGGTGT
T Ding Ding TTGMAGAG TCGTGAAGCG
GCTGGCGCGA GAGGCGCCTG
GCATCGGCTTCGAGTTGCGCCTCT
CACTGAGGA DingCCCGATGAACTTCTC
CGGTACGGTGACGTCGATTTCGTGA
TCC ding T CCGGMTTTGT TCGCGTC
GAG CGATCATCCA AAGGCGAA
AC TGCTCGACGACACGCTGGn T
GCGTAGGTT GCCCCACTAA CAAG
CAGTTA AAACGGCAGC TTTCTTT
CGAAAAC Ding ACGGATCGA Ding GGG Ding CAT
ATTGCGGCTAAGTTCGGACGTACGC
TMAGCCCTCCATCGAGMTTG TrG
TTGCTTG AGCACGGTCT CAAGAG
GCGC ATCGAAGTCG TCGTGCCG
GATTTAGTCTA ATCCCGCCTT
TGCTGTCGGG AACCGA CGC A
TAGCGACCA TGCCGTTACGCTGG
ATGCGG CAGGTAC TINGAC TACAG
GAAGC G-TGCACATG ACAACT
CCGC GTGA DINGTCAGTGGMTATCGA
CCCTAGTCCGding TCGCAGCCGC
GGATTTGACC GGCTT Ding GAGA C
ACCCTGCTCACGA Ding CAATG TGCC
GCCCCG CCATGA Ding CA Ding GMGCAA
GAC CCTGGTACAC GGCTTCGG
CCGCGGAGTGGG CnTTGTGTG 60
1 & 3 K Sv A
(:DRI Z D cF
MVQRRVD! V KLGIG
LτED Y V LAS'
l) KLDTL C KOKR F
E(; IKRTLKIN L KZ
V SL LM VE TT
t/A. LALPAQ SLNRQI
(7/t. L upper lli: EAl4-1
7 Tongue = 3-a Eni/Fk No. A-Ala = Alanine (,41Qr++ha)
E = GI Tsu・tsu゛1re+E 冫内t ('q(,.1
%-ic AciJ冫H=HfsszSu-'}”;7(
Hr5shi-cl;ncku18I1e8 7-leucine
(engine soleucIna) κ= Lys Tomi so;
'7 (Ly61pe- )L=Leu s Leucjhe M=Met= 7/+Saiz-/ CHt6'+one )N-Asn w
Ryozu n"5 keys" (Aspara5rhc pt.
= Pro = ao'l冫('Pr.l1'ha
)Q = G]n ” /7-izzE 7 ((z
l. a+arrnhe)R=Arg”'i
't Rekin (Art31vsjse)S''
S"'"u'ln(Ser'+ne)T"
Thr”7ReTz7 (%reoMne)V
= Vat "1)"ri7 (1/ali
na ) W = Try ・ Tosoatomanton (Trpifo will come true Y = Tyr = 4 mouths of medicine ζ 7yros
i n e. Figure 1 is a partial restriction map of the two Eco RI fragments.

that's all

Claims (1)

[Claims] 1. Rhizobium japonicum
￥￥japonicum￥）USDA191￥no
Cloned DNA containing a d\D-related gene or a functionally equivalent homolog thereof. 2. The \nod\D-related gene is \nod\D-r1
, \nod\D-r2 or a functionally equivalent homolog thereof. 3. The \nod\D-related gene is \nod\D-r1
Or the first claim which is ¥nod¥D-r2
DNA described in section. 4. Claim 1, wherein the DNA is a plasmid
DNA described in section. 5. The DNA is Rhizobium (￥Rhizobium￥
) The DNA according to claim 1, which is a plasmid that can replicate in multiple copies within a host. 6. Rhizobium japonicum
￥￥japonicum￥）USDA191￥no
A cloned DNA containing a structural gene encoding the amino acid sequence of a d\D-related protein or a homolog functionally equivalent to the protein. 7. The structural gene is \nod\D-r1, \nod\
The DNA according to claim 6, which is D-r2 or a functionally equivalent homolog thereof. 8. The structural gene is \nod\D-r1 or \n
The cloned DNA according to claim 6, which is od\D-r2. 9. Claim 6, wherein the DNA is a plasmid.
Cloned DNA as described in Section. 10. The DNA is Rhizobium
¥) The DNA according to claim 6, which is a plasmid that can replicate in multiple copies within a host. 11. A promoter of \nod\D-r1, \nod\D-r2, or a functionally equivalent homolog having at least 90% sequence homology thereto, which is combined with an exogenous coding sequence and the combination is Rhizobium (¥Rhiz
o-bium\) A promoter that can be expressed within the host. 12, ¥nod¥D-r1 or ¥nod¥D-r2
12. The promoter according to claim 11, containing the promoter of claim 11. 13. Claim 11 contained within the plasmid
Promoters as described in Section. 14. The promoter according to claim 11, which is contained in a plasmid that can replicate in multiple copies in a Rhizobium host.